New Developments in Immunochemistry with Immunoalkaline Phosphatase Methods

Yam, Lung T.; Khansur, Tawfiq; Tavassoli, Mehdi

doi:10.1159/000157114

Cited by 6 publications

(5 citation statements)

References 30 publications

(51 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Alkaline phosphatase conjugates are also common amplification reagents due to the orthogonality of the high pH conditions to endogenous enzymes and the availability of fluorescent and colorimetric substrates. 156,157 Using a similar strategy to the β-galactosidase assay described above (Figure 13C), Rana et al 158 synthesized an alkaline phosphatase substrate containing a masked quinone methide-tellurophene functionality (QM-Te, Figure 15A). This substrate was benchmarked against a Maxpar reagent carrying 175 Lu in an IMC experiment against PDL1 expressing cell cores (Figure 15B).…”

Section: Signal Amplificationmentioning

confidence: 99%

“…They are traditionally used in immunohistochemistry in a technique commonly referred to as brown staining, which uses Ab-horseradish peroxidase conjugates. Alkaline phosphatase conjugates are also common amplification reagents due to the orthogonality of the high pH conditions to endogenous enzymes and the availability of fluorescent and colorimetric substrates. , …”

Section: Small Molecule Mass Tag Reagentsmentioning

confidence: 99%

See 1 more Smart Citation

Reagents for Mass Cytometry

et al. 2023

View full text Add to dashboard Cite

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

show abstract

Section: Signal Amplificationmentioning

confidence: 99%

Section: Small Molecule Mass Tag Reagentsmentioning

confidence: 99%

Reagents for Mass Cytometry

et al. 2023

View full text Add to dashboard Cite

show abstract

“…We proposed a single QM precursor scaffold containing an amine-functionalized linker group allowing for simple conjugation to nearly any reporter molecule. Given our primary application of CARD for solid-phase immunoassays such as IHC on FFPE tissue, the use of the phosphate group as the enzyme-cleavable recognition head was a logical choice due to the ubiquity of its cognate enzyme AP in current immunoassays . The application of AP-based CARD for IHC begins with the incubation of a primary antibody (Ab) that recognizes the antigen of interest, followed by incubation with a secondary Ab:AP complex that binds the primary Ab by typical antispecies Ab binding (Figure ).…”

Section: Introductionmentioning

confidence: 99%

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates

et al. 2016

View full text Add to dashboard Cite

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.

show abstract

“…AP-conjugates are ubiquitous in immunoassays with numerous well-characterized synthetic substrates. , Latent quinone methide (QM) derivatives, developed originally as AP covalent inhibitors, , have been used to covalently label phosphatases in solution for visualization. − Kinetic analysis and product analysis of QMs generated enzymatically suggest these products diffuse away from the enzymes active site and label local nucleophiles. , In the case of an AP antibody conjugate, the QM released will be immobilized by local nucleophiles, in close proximity to the site of QM generation (Scheme ). Provided adequate turnover can be realized, a significant degree of signal amplification with minimal diffusion can be achieved.…”

mentioning

confidence: 99%

“…AP-conjugates are ubiquitous in immunoassays with numerous well-characterized synthetic substrates. 18,19 Latent quinone methide (QM) derivatives, developed originally as AP covalent inhibitors, 20,21 have been used to covalently label phosphatases in solution for visualization. 22−26 Kinetic analysis and product analysis of QMs generated enzymatically suggest these products diffuse away from the enzymes active site and label local nucleophiles.…”

mentioning

confidence: 99%

Signal Amplification for Imaging Mass Cytometry

et al. 2019

View full text Add to dashboard Cite

An enzyme-catalyzed reporter deposition stain has been developed for Imaging Mass Cytometry (IMC). The reagent consists of an alkaline phosphatase substrate tethered to a tellurophene which serves as reporter group for mass cytometry. Upon phosphate hydrolysis, a quinone methide is released which covalently labels local nucleophiles. This strategy is a useful complement to heavy isotope antibody conjugates as it facilitates signal amplification for low-abundance biomarker detection. The workflow is conveniently integrated with standard IMC antibody staining to allow multiparametric antigen detection.

show abstract

New Developments in Immunochemistry with Immunoalkaline Phosphatase Methods

Cited by 6 publications

References 30 publications

Reagents for Mass Cytometry

Reagents for Mass Cytometry

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates

Signal Amplification for Imaging Mass Cytometry

Contact Info

Product

Resources

About